Cytotoxic agents for the treatment of cancer

ABSTRACT

The present invention relates to a method of treating brain cancer comprising administering a therapeutically effective substance to a patient, wherein the therapeutically effective substance comprises: (I), or a pharmaceutically acceptable salt thereof, wherein X is a moiety that can be cleaved hydrolytically or enzymatically in the body of the patient in a pH-dependent manner.

CROSS REFERENCE TO RELATED APPLICATIONS

This application is a continuation of U.S. application Ser. No.14/893,821, filed Nov. 24, 2015, now U.S. Pat. No. 10,278,981, which isa national stage application under 35 U.S.C. 371 and claims the benefitof PCT Application No. PCT/US2014/040872 having an international filingdate of Jun. 4, 2014, which designated the United States, which PCTapplication claimed the benefit of U.S. Provisional Application No.61/831,219, filed Jun. 5, 2013, the disclosures of each of which areherein incorporated by reference in their entireties.

BACKGROUND OF THE INVENTION

Anthracyclines are a class of antibiotics derived from certain types ofStreptomyces bacteria. Anthracyclines are often used as cancertherapeutics and function in part as nucleic acid intercalating agentsand inhibitors of the DNA repair enzyme topoisomerase II, therebydamaging nucleic acids in cancer cells, preventing the cells fromreplicating. One example of an anthracycline cancer therapeutic isdoxorubicin, which is used to treat a variety of cancers includingbreast cancer, lung cancer, ovarian cancer, lymphoma, and leukemia. The6-maleimidocaproyl hydrazone of doxorubicin (DOXO-EMCH also known asaldoxorubicin or INNO-206) was synthesized to provide an acid-sensitivelinker that could be used to prepare immunoconjugates of doxorubicin andmonoclonal antibodies directed against tumor antigens (Willner et al.,Bioconjugate Chem 4:521-527 (1993)). In this context, antibody disulfidebonds are reduced with dithiothreitol to form free thiol groups, whichin turn react with the maleimide group of DOXO-EMCH to form a stablethioether bond. When administered, the doxorubicin-antibody conjugate istargeted to tumors containing the antigen recognized by the antibody.Following antigen-antibody binding, the conjugate is internalized withinthe tumor cell and transported to lysosomes. In the acidic lysosomalenvironment, doxorubicin is released from the conjugate intracellularlyby hydrolysis of the acid-sensitive hydrazone linker. Upon release, thedoxorubicin reaches the cell nucleus and is able to kill the tumor cell.For additional description of doxorubicin and DOXO-EMCH see, forexample, U.S. Pat. Nos. 7,387,771 and 7,902,144 and U.S. patentapplication Ser. No. 12/619,161, each of which is incorporated in theirentirety herein by reference.

Further, DOXO-EMCH has been conjugated in vitro with human serum albumin(HSA) to form a stable thioether conjugate (Kratz et al., J Med. Chem45:5523-5533 (2002)).

Brain tumors, including malignant gliomas in particular, are among themost aggressive human cancers and are rarely curable (DeAngelis et al.,N. Engl. J. Med. 2001, 344, 114-123; Nelson et al., J. Neurooncol. 1985,3, 99-103; Kornblith et al., J. Neurosurg. 1988, 68, 1-17). The mediansurvival after diagnosis is about 12-14 months. Treatment withchemotherapeutic drugs such as nitrosoureas, platinum compounds, andtemozolomide, increases the survival time of patients only marginally(Huncharek et al., Anticancer Res. 1998, 18, 4693-4697; Brandes et al.,Anticancer RES. 2000, 20, 1913-1920). Further complicating treatment ofbrain cancers is the inability of many drugs to cross the blood-brainbarrier (BBB). The BBB consists of tight junctions around thecapillaries and protect the brain against changes in the levels ofcertain substances like ions, or against infections. The endothelialcells restrict the diffusion of large molecules such as albumin, whileallowing the diffusion of smaller molecules such as O₂ or CO₂.Glioblastoma cell lines were driven to apoptosis following growth arrestinduced by doxorubicin (Stan et al., Anticancer Res., 1999, 19,941-950). However, doxorubicin lacks the ability to cross the BBB,because it is a substrate of the P-glycoprotein efflux pumps (Sardi etal., Cancer Chemother. Pharmacol., 2011, 67, 1333-1340). Freedoxorubicin concentration in glioma tissues is below effective levelsand doxorubicin has no effect on glioblastoma growing in the brain(Steiniger et al., Int. J. Cancer, 2004, 109, 759-767; Von Hoist, ActaNeurochirr., 1990, 104, 13-16). Therefore, the development of a strategyallowing drug delivery across the BBB is of prime importance. Hence, theneed for efficient carriers to transport anticancer drugs, such asdoxorubicin, into the brain remains high.

SUMMARY OF THE INVENTION

The present invention is based on the surprising observation that whenadministered intravenously, a therapeutically effective substance (e.g.,DOXO-EMCH) induces tumor regression in and significantly increasessurvival of a mammal suffering from a brain tumor such as e.g.,glioblastoma multiforme tumors.

The present invention relates to a method for the treatment of braincancer comprising administering a therapeutically effective substance toa patient, wherein the therapeutically effective substance comprises thestructure as in FIG. 13, or a pharmaceutically acceptable salt thereofwherein X is a bond that can be cleaved hydrolytically or enzymaticallyin the body of the patient in a pH-dependent manner.

In some embodiments, the moiety X is cleaved in the body of the patient,thereby releasing the cytotoxic agent. In some embodiments, thecytotoxic agent is an anthracycline. In some embodiments, theanthracycline is selected from a group consisting of doxorubicin,daunorubicin, epirubicin, idarubicin, valrubicin, pirarubicin,zorubicin, aclarubicin, caminomycin, mitoxantrone and ametantrone, or aderivative of any of the foregoing, or a pharmaceutically acceptablesalt of any of the foregoing. In some embodiments, the anthracycline isdoxorubicin or a pharmaceutically acceptable salt thereof. In someembodiments, the covalently protein-binding group is selected from thegroup consisting of maleimide, haloacetamide, haloacetate, pyridylthio,N-hydroxysuccinimide ester, isothiocyanate, disulfide, vinylcarbonyl,aziridine and acetylene. In some embodiments, the covalentlyprotein-binding group is maleimide. In some embodiments, the linkercomprises an organic molecular residue, which contains at least onealiphatic carbon chain, or an aliphatic carbon ring having 1-12 carbonatoms, some of which can be replaced with heteroatoms, or an aromaticmoiety. In some embodiments, the linker comprises at least one carbonchain having 1-12 carbon atoms. In some embodiments, the cytotoxic agentand the linker are joined by a hydrazone moiety. In some embodiments,the therapeutically effective substance has the following structure:

In some embodiments, the brain cancer is a primary brain cancer. In someembodiments, the primary brain cancer is glioma, astrocytoma,oligodendroglioma, ependymoma, meningioma, craniopharyngioma, germinoma,pineocytoma, pineoblastoma and glioblastoma multiforme. In someembodiments, the primary brain cancer is glioblastoma multiforme. Insome embodiments, the brain cancer is a secondary or metastatic cancer.In some embodiments, the secondary or metastatic cancer is selected frombladder cancer, breast cancer, lung cancer, stomach cancer, endometrialcancer, ovarian cancer, pancreatic cancer, pancreatic ductaladenocarcinoma, cancer of the adrenal cortex, non-Hodgkin's lymphoma,multiple myeloma, leukemia, Kaposi's sarcoma, Ewing's sarcoma, softtissue sarcoma, nephroblastoma, prostate cancer, liver cancer, bonecancer, chondrosarcoma, renal cancer, bladder cancer, thyroid cancer andgastric cancer. In some embodiments, the cancer is atemozolomide-resistant cancer. In some embodiments, thetemozolomide-resistant cancer is a temozolomide-resistant glioblastomamultiforme.

In some embodiments, the therapeutically effective substance isadministered in combination with an anti-cancer agent. In someembodiments, the anti-cancer agent is selected from doxorubicin,cisplatin, carboplatin, paclitaxel, docetaxel, temozolomide,nitrosoureas, bortezomib, gemcitabine, etoposide, topotecan, or apharmaceutically acceptable salt thereof.

Upon administration, the therapeutically effective substance bindscovalently, by way of the protein-binding molecule, to body fluidconstituents or tissue constituents, thereby creating a transport formof the cytotoxic agent which can be hydrolytically or enzymaticallycleaved, in a pH-dependent manner, in the body with the cytotoxic agentbeing released. Because of their protein-binding properties, injectablemedicament preparations are obtained of therapeutically effectivesubstances that decisively alter and improve the pharmacokinetic profileof the cytotoxic agent. When the therapeutically effective substance ofthe invention interacts with body fluids, it binds covalently to bodyfluid or tissue constituents, preferably to serum proteins, morepreferably to serum albumin, in order to yield macromolecular prodrugswhich transport the cytotoxic agent to the target site and/or release itin a metered form.

In some embodiments, the brain cancer is a primary brain tumor. In someembodiments, the primary brain tumor is glioblastoma multiforme. Inother embodiments, the brain cancer is a metastatic brain tumor. In someembodiments, the metastatic tumor is from a cancer including but notlimited to breast cancer, lung cancer, stomach cancer, endometrialcancer, ovarian cancer, pancreatic cancer, pancreatic ductaladenocarcinoma, cancer of the adrenal cortex, non-Hodgkin's lymphoma,multiple myeloma, leukemia, Kaposi's sarcoma, Ewing's sarcoma, softtissue sarcoma, nephroblastoma, glioblastoma, prostate cancer, livercancer, bone cancer, chondrosarcoma, renal cancer, bladder cancer,thyroid cancer and gastric cancer. In some embodiments, the tumor is atemozolomide-resistant tumor. In some embodiments, thetemozolomide-resistant tumor is a temozolomide-resistant glioblastomamultiforme.

In some embodiments, the therapeutically effective substance is used inthe manufacture of a medicament for the treatment of brain cancer suchas e.g., glioblastoma multiforme. In some embodiments, the inventionprovides a therapeutically effective substance for use in the treatmentof brain cancer in a patient. In some embodiments, the inventionprovides a therapeutically effective substance for use in the treatmentof glioblastoma multiforme in a patient. In some embodiments, theinvention provides a therapeutically effective substance for thetreatment of brain cancer in a patient. In some embodiments, theinvention provides a therapeutically effective substance for thetreatment of brain cancer in a patient.

BRIEF DESCRIPTION OF THE FIGURES

FIG. 1 shows that aldoxorubicin reduces glioblastoma multiforme (GBM)tumor burden in a murine model. Female Balb/c (nu/nu) mice wereimplanted with 5×10⁵ firefly luciferase-labeled U87MG (U87-luc) gliomacells intracranially on day 0. After 12 days, control mice in group C(1-8) received tail vein injections of drug vehicle, while mice in thetreatment group D (9-16) were injected with 120 μg/injection ofdoxorubicin (based on a 20 g body weight), and mice in the treatmentgroup A (17-24) received 480 μg/inj of aldoxorubicin. Injection withdoxorubicin was repeated after 19 days and with aldoxorubicin after 19and 26 days. Bioluminescence imaging of brain tumors was performed after8, 16, 22, 27, 34, and 41 days of tumor cell implantation, and is shownas a function of photon/sec/cm²/sr in each picture (the radiance unit ofphotons/sec/cm²/sr is the number of photons per second that leave asquare centimeter of tissue and radiate into a solid angle of onesteradian (sr)). Tumor burden is demonstrated through a colorimetricscale as shown, where the highlighted areas represent the greatestsignal intensity (highest tumor burden). \ indicates death of theanimal.

FIG. 2 shows scatter plots for mice in control (C) and treatment groupsdoxorubicin (D) and aldoxorubicin (A) displaying the relationshipbetween tumor sizes expressed as a function of radiance(photons/sec/cm²/sr) obtained from images shown in FIG. 1. There was norelative difference in average tumor sizes between the control and thetreatment groups after 8 days of cell implantation (p>0.05) (compareFigure A, B, and C). Difference between the control group and thealdoxorubicin group, but not the doxorubicin group, was observed 22 daysafter implantation (p=0.005; shown by an asterisk, FIG. 2D). There wasno relative difference in tumor growth between 8 days and 22 days in thealdoxorubicin group (p>0.05) (FIG. 2C). However, a relative differencein tumor growth between 8 and 22 days was observed in the doxorubicingroup (p<0.05) (FIG. 2B), indicating suppression of tumor growth byaldoxorubicin but not doxorubicin.

FIG. 3 shows Kaplan-Meier survival curves by days of study forGBM-bearing mice. Mice receiving aldoxorubicin treatment (n=8; solidline (d)) survived a longer time (p≤0.0001) than mice receiving vehicle(dashed line labelled (c)) or doxorubicin (dashed line labelled (d)).There was no difference in the survival curves between vehicle-treatedand doxorubicin-treated mice (p=0.949). As described herein,aldoxorubicin was administered intravenously for a total of sixinjections (i.e., 12, 19, 26, 42, 50, and 56 days after cellimplantation). All the doses were ˜75% of the maximum tolerated dose(MTD) of 32 mg/kg/injection, except that the dose given after 50 days ofcell implantation was 50% of the MTD. Doxorubicin was administered for atotal of two injections (i.e., 12 and 19 days after cell implantation)with ˜75% of the MTD of 8 mg/kg/injection.

FIG. 4 shows the concentration-time profile of aldoxorubicin in varioustissues following intravenous administration of 24 mg/kg dose (75% ofthe maximum tolerated dose) to intracranial GBM tumor-bearing mice.Lines represent the mean data (ng of aldoxorubicin/ml of plasma ortissues extracts) obtained from three mice sampled at each time point.Bars indicate ±standard deviation. Aldoxorubicin concentrations in brainrepresent values obtained from total brain tissues from tumor-bearingmice.

FIG. 5 shows luciferase expression levels in GBM tumors implantedintracranially into mice. Implanted human GBM tumor cells containing theluciferase gene were allowed to grow in the brains of mice for 9 daysprior to treatment with either buffer control (C) or aldoxorubicin (T).Mice were administered either buffer (C) or aldoxorubicin on Days 9, 16and 23. Tumor growth was monitored by detecting bioluminescence ofluciferase substrate administered to mice prior to scanning on Days 7,21, 29 and 33. Intensity of luciferase expression in tumor cells isshown according to the color scheme on the right, with the darkenedareas indicating greatest expression (most tumor cells) and blackindicating no expression (no tumor cells).

At 7 days, tumor cell expression of luciferase was seen in both control(C) and aldoxorubicin-treated (T) animals. At 21 days, all control micehad growing tumors, while only 1 aldoxorubicin-treated mouse haddetectable tumor. At Day 29, all C mice had died, while only 1aldoxorubicin-treated mouse was dead and tumor regrowth was observed in2 mice. At Day 33, 3 T mice had died and tumor had regrown in 4 othermice. † indicates death of the animal.

FIG. 6 shows scatter plots for mice in control (C) and aldoxorubicintreatment groups (T) displaying the relationship between tumor sizesexpressed as a function of radiance (photons/sec/cm²/sr) obtained fromimages shown in FIG. 5. There was no relative difference in averagetumor sizes between the control group and the treatment group after 7days (p>0.05) (FIG. 6A), but differences between the control group andthe treatment groups after 21 days were observed (p<0.05, see asteriskin FIG. 6B). There was no relative difference in the treatment groupsbetween 21 days to 29 days (p>0.05), but difference was observed at 33days as a result of the reappearance of the tumors (p<0.05; shown by anasterisk in FIG. 6C).

FIG. 7 shows that Kaplan-Meier survival curves by days of study forGBM-bearing mice. Mice receiving aldoxorubicin (n=8; dashed line)survived longer (p=0.0006) than those receiving vehicle (n=8: solidline).

FIG. 8 shows that aldoxorubicin retention was 3- to 4-fold higher intumor tissues than in the surrounding brain tissues. Aldoxorubicinretention in tumors and surrounding brain tissues of mice was measuredby HPLC following intravenous administration of 24 mg/kg dose (75% ofthe maximum tolerated dose) to intracranial GBM tumor-bearing mice.

FIG. 9 shows a graphical representation of the immunohistochemicalevaluation of aldoxorubicin (ALDOX) treatment on the proliferation(Ki-67), intratumoral vasculature (CD31), intermediate filament proteinexpression (vimentin), and activation of apoptosis effector (cleavedcaspase-3), versus control (NT).

FIG. 10 shows selective accumulation of aldoxorubicin but notdoxorubicin in the intracranial human glioblastoma tumors in athymicnude mice. Tumor-bearing mice received intravenous injections ofaldoxorubicin and doxorubicin as described in Example 1. Mice wereeuthanized 24 h following the last injection. Brains were harvested andimaged using a stereomicroscope equipped for brightfield andepiflorescence at doxorubicin-specific wavelengths to visualize drugaccumulation.

FIG. 11 shows selective accumulation of aldoxorubicin but notdoxorubicin as seen in the cryosections of the brain tissues oftumor-bearing mice. Immunohistochemical staining for CD31, amicro-vessel density marker, and Vimentin, a proinvasive type IIIintermediate filament protein is also shown. All nuclei werecounterstained with DAPI.

FIG. 12 shows bar graphs for mice in control (control), doxorubicin(doxo), and aldoxorubicin (aldoxo) treatment groups displaying therelationship between tumor sizes expressed as a function of radiance(photons/sec/cm²/sr) obtained from images shown in FIG. 1 and time.

FIG. 13 shows an illustration of the therapeutically effective substanceuseful in the present invention. X is a bond that can be cleavedhydrolytically or enzymatically in the body of a patient in apH-dependent manner.

DETAILED DESCRIPTION OF THE INVENTION

Unless otherwise defined herein, scientific and technical terms used inthis application shall have the meanings that are commonly understood bythose of ordinary skill in the art. Generally, nomenclature used inconnection with, and techniques of, chemistry, molecular biology, celland cancer biology, immunology, microbiology, pharmacology, and proteinand nucleic acid chemistry, described herein, are those well known andcommonly used in the art.

All publications, patents and published patent applications referred toin this application are specifically incorporated by reference herein.In case of conflict, the present specification, including its specificdefinitions, will control. Unless otherwise specified, it is to beunderstood that each embodiment of the invention may be used alone or incombination with any one or more other embodiments of the invention.

Throughout this specification, the word “comprise” or variations such as“comprises” or “comprising” will be understood to imply the inclusion ofa stated integer (or components) or group of integers (or components),but not the exclusion of any other integer (or components) or group ofintegers (or components).

The singular forms “a,” “an,” and “the” include the plurals unless thecontext clearly dictates otherwise.

The term “including” is used to mean “including but not limited to.”“Including” and “including but not limited to” are used interchangeably.

For a number qualified by the term “about,” a variance of 2%, 5% or even10% is within the scope of the qualified number.

The term “anthracycline” refers to a class of antineoplastic antibioticshaving an anthracenedione (also termed anthraquinone or dioxoanthracene)structural unit. For example, the term “anthracycline” is specificallyintended to individually include doxorubicin, daunorubicin, epirubicin,idarubicin, valrubicin, pirarubicin, zorubicin, aclarubicin,caminomycin, mitoxantrone, and ametantrone.

The terms “patient” and “individual” are used interchangeably and referto either a human or a non-human animal. These terms include mammalssuch as humans, primates, livestock animals (e.g., bovines, porcines),companion animals (e.g., canines, felines) and rodents (e.g., mice andrats).

The term “pharmaceutically acceptable” means a non-toxic material thatdoes not interfere with the effectiveness of the biological activity ofthe active ingredient(s). The characteristics of the carrier will dependon the route of administration.

The term “pharmaceutically acceptable carrier” refers to a non-toxiccarrier that may be administered to a patient, together with atherapeutically effective substance of this invention, and which doesnot destroy the pharmacological activity of the cytotoxic agent. Theterm “excipient” refers to an additive in a formulation or compositionthat is not a pharmaceutically active ingredient.

The term “pharmaceutically effective amount” or “therapeuticallyeffective amount” refers to an amount effective to treat brain cancer ina patient, e.g., effecting a beneficial and/or desirable alteration inthe general health of a patient suffering from a disease (e.g., cancer).The skilled worker will recognize that treating brain cancer includes,but is not limited to, killing cancer cells, preventing the growth ofnew cancer cells, causing tumor regression (a decrease in tumor size),causing a decrease in metastasis, improving vital functions of apatient, improving the well-being of the patient, decreasing pain,improving appetite, improving the patient's weight, and any combinationthereof. A “pharmaceutically effective amount” or “therapeuticallyeffective amount” also refers to the amount required to improve theclinical symptoms of a patient. The therapeutic methods or methods oftreating brain cancer described herein are not to be interpreted orotherwise limited to “curing” brain cancer.

As used herein, the term “treating” or “treatment” includes reversing,reducing, or arresting the symptoms, clinical signs, and underlyingpathology of a condition in manner to improve or stabilize a subject'scondition. As used herein, and as well understood in the art,“treatment” is an approach for obtaining beneficial or desired results,including clinical results. Beneficial or desired clinical results caninclude, but are not limited to, alleviation or amelioration of one ormore symptoms or conditions, diminishment of extent of disease,stabilized (i.e., not worsening) state of disease, delay or slowing ofdisease progression, amelioration or palliation of the disease state,and remission (whether partial or total), whether detectable orundetectable. “Treatment” can also mean prolonging survival as comparedto expected survival if not receiving treatment.

The term “substituted” refers to moieties having substituents replacinga hydrogen on one or more carbons of the backbone of a chemicalcompound. It will be understood that “substitution” or “substitutedwith” includes the implicit proviso that such substitution is inaccordance with permitted valence of the substituted atom and thesubstituent, and that the substitution results in a stable compound,e.g., which does not spontaneously undergo transformation such as byrearrangement, cyclization, elimination, etc. As used herein, the term“substituted” is contemplated to include all permissible substituents oforganic compounds. In a broad aspect, the permissible substituentsinclude acyclic and cyclic, branched and unbranched, carbocyclic andheterocyclic, aromatic and non-aromatic substituents of organiccompounds. The permissible substituents can be one or more and the sameor different for appropriate organic compounds. For purposes of theinvention, the heteroatoms such as nitrogen may have hydrogensubstituents and/or any permissible substituents of organic compoundsdescribed herein which satisfy the valences of the heteroatoms.Substituents can include any substituents described herein, for example,a halogen, a hydroxyl, a carbonyl (such as a carboxyl, analkoxycarbonyl, a formyl, or an acyl), a thiocarbonyl (such as athioester, a thioacetate, or a thioformate), an alkoxyl, an alkylthio,an acyloxy, a phosphoryl, a phosphate, a phosphonate, an amino, anamido, an amidine, an imine, a cyano, a nitro, an azido, a sulfhydryl,an alkylthio, a sulfate, a sulfonate, a sulfamoyl, a sulfonamido, asulfonyl, a heterocyclyl, an aralkyl, or an aromatic or heteroaromaticmoiety.

Therapeutically Effective Substance

The therapeutically effective substance useful in the present inventioncomprises the structure of FIG. 13.

-   or a pharmaceutically acceptable salt thereof,-   wherein X is a bond that can be cleaved hydrolytically or    enzymatically in the body of the patient in a pH-dependent manner.

In some embodiments, the cytotoxic agent used in the therapeuticallyeffective substance is an anthracycline. Anthracyclines include, but arenot limited to, doxorubicin, daunorubicin, epirubicin, idarubicin,valrubicin, pirarubicin, zorubicin, aclarubicin, caminomycin,mitoxantrone, and ametantrone, or a derivative thereof. In someembodiments, the anthracycline is doxorubicin or a pharmaceuticallyacceptable salt, thereof.

In some embodiments, the cleavable moiety (“X”) is an acid-cleavablemoiety. Acid-cleavable moieties include, but are not limited to, acetal,ketal, imine, hydrazone, carboxylhydrazone or sulphonylhydrazone, orcis-aconityl moieties or moieties containing a substituted orunsubstituted trityl group. In certain embodiments, the acid-cleavablemoiety is a hydrazone moiety. In some embodiments, the cytotoxic agentis released when moiety X is cleaved in the body of the patient.

In some embodiments, the cleavable moiety (“X”) is enzyme-cleavable.Enzyme-cleavable moieties include, but are not limited to, peptidecomprising one or more carbamate bonds. A peptide moiety may comprise,for example, 1-5, 1-10, 1-15, 1-20, 1-25, 1-30, 1-35, 1-40, 2-5, 2-10,2-15, 2-20, 2-25, 2-30, 2-35, or 2-40 amino acid residues. Peptidemoieties include, but are not limited to, moieties comprising 1, 2, 3,4, 5, 6, 7, 8, 9, 10, 11, 12, 13, 14, 15, 16, 17, 18, 19, 20, 21, 22,23, 24, 25, 26, 27, 28, 29, 30, 31, 32, 33, 34, 35, 36, 37, 38, 39, or40 amino acid residues. A peptide moiety may be designed to bespecifically cleavable by one or more proteases. In some embodiments,the bond being cleaved is a peptide bond, an imide bond, or acarboxyl-hydrazone bond of a hydrazine moiety.

In some embodiments, the linker is an organic molecule. Such linker maycomprise at least one aliphatic carbon chain and/or an aliphatic carbonring with 1-12 carbon atoms, wherein any of the carbon atoms may besubstituted with an —OH or ═O, and wherein any of the carbon atoms maybe replaced with heteroatoms or an aromatic moiety where appropriate andchemically feasible. In some embodiments, the heteroatom is oxygen. Insome embodiments, the aliphatic linker may comprise an alkyl chaincomprising 1-12 carbon atoms, an alkenyl chain comprising 2-12 carbonatoms, or an alkynyl chain comprising 2-12 carbon atoms, wherein any ofthe carbon atoms maybe substituted with an —OH or ═O, and wherein any ofthe carbon atoms may be replaced with oxygen atoms where appropriate andchemically feasible. In particular embodiments, the aliphatic linker isan alkyl chain comprising 1-12 carbon atoms, wherein any of the carbonatoms maybe substituted with an ═O, where appropriate and chemicallyfeasible. In yet other embodiments, the aliphatic linker is an alkylchain comprising 3-9, 4-8, or 5-7 carbon atoms, wherein any of thecarbon atoms maybe substituted with an ═O, where appropriate andchemically feasible. In a particular embodiment, the aliphatic linker isan alkyl chain comprising 6 carbon atoms, wherein the carbon atomattached to the cleavable moiety “X” is substituted with an ═O.

The covalently protein-binding group include, but is not limited to, amaleimide group, a haloacetamide group, a haloacetate group, apyridyldithio group, an N-hydroxysuccinimide ester group, and anisothiocyanate group. In certain embodiments, the covalentlyprotein-binding group is a maleimide group. Covalently protein-bindinggroups also include a disulfide group, a vinylcarbonyl group, anaziridine group or an acetylene group. A disulfide group may beactivated by a thionitrobenzoic acid (e.g. 5′-thio-2-nitrobenzoic acid)as the exchangeable group. A maleimide, pyridyldithio, orN-hydroxysuccinimide ester group can, where appropriate, be substitutedby an alkyl group or by the above water-soluble groups. In general, aprotein-binding group possesses protein-binding properties, i.e., itbinds covalently (“a covalent protein-binding group”) or noncovalently(“a noncovalent protein-binding group”), in a physiological environment,to particular amino acids on the surface of the protein. The maleimidegroup, the haloacetamide group, the haloacetate group, the pyridyldithiogroup, the disulfide group, the vinylcarbonyl group, the aziridinegroup, and/or the acetylene group preferably reacts with thiol (—SH)groups of cysteines, while the N-hydroxysuccinimide ester group and/orthe isothiocyanate group preferably react with the amino group (—NH) oflysines, on the surface of a protein. For example, the covalentlyprotein-binding group (such as a maleimide group) may bind to albumin.In some embodiments, the albumin is not modified (e.g., it is notmodified to be charged, either positively or negatively).

The therapeutically effective substance used in the invention includesany and all combinations of one or more anthracyclines, cleavablemoieties, linkers, and covalently protein-binding groups.Therapeutically effective substances may comprise an anthracycline, anacid-cleavable moiety, an alkyl linker, and a covalently protein-bindinggroup. In certain embodiments, the therapeutically effective substancecomprises an anthracycline, a hydrazone as the acid-cleavable moiety, analkyl linker, and a maleimide group as the covalently protein-bindinggroup. In other embodiments, the therapeutically effective substancecomprises an anthracycline, a hydrazone moiety as the acid-cleavablemoiety, a 6-carbon alkyl linker wherein the carbon atom attached to thecleavable moiety is substituted with an ═O, and a maleimide group as thecovalently protein-binding group (i.e., an anthracycline-EMCH molecule).

An exemplary compound used in the present invention is DOXO-EMCH. Theterm “DOXO-EMCH,” alone or in combination with any other term, refers toa compound as depicted by the following structure:

DOXO-EMCH is also referred to as(E)-N′-(1-((2S,4S)-4-(4-amino-5-hydroxy-6-methyl-tetrahydro-2H-pyran-2-yloxy-2,5,12-trihydroxy-7-methoxy-6,11-dioxo1,2,3,4,6,11-hexahydrotetracen-2-yl)-2-hydroxyethylidene)-6-(2,5-dioxo-2H-pyrrol-1(5H)yl)hexanehydrazide.HCl.Pharmaceutical Compositions

In some embodiments, the invention provides a pharmaceutical compositionfor use in the treatment of brain cancer in a patient comprising atherapeutically effective substance, wherein the therapeuticallyeffective substance comprises FIG. 13, or a pharmaceutically acceptablesalt thereof wherein X is a moiety that can be cleaved hydrolytically orenzymatically in the body of the patient in a pH-dependent manner.

Each of the methods or uses of the present invention, as describedherein, comprises administering to a patient a therapeutically effectivesubstance or a pharmaceutically acceptable salt or ester form thereof totreat brain cancer. In some embodiments, the therapeutically effectivesubstance may be administered alone. In some embodiments, thetherapeutically effective substance may be administered in combinationwith an anti-cancer agent. In some embodiments, the therapeuticallyeffective substance may be administered in combination with othermedications such as the anthracyclines, platinum-containing anti-cancercompounds, taxanes, alkylating agents, proteasome inhibitors, nucleosideanalogs, topoisomerase inhibitors, immunosuppressive agents for thetreatment of immune-mediated brain disorders. In some embodiments, thetherapeutically effective substance may be administered in combinationwith other medications such as doxorubicin, cisplatin, carboplatin,paclitaxel, docetaxel, temozolomide, nitrosoureas, bortezomib,gemcitabine, etoposide, topotecan, or a pharmaceutically acceptable saltthereof.

The total amount of a therapeutically effective substance (e.g.,DOXO-EMCH) in a composition to be administered to a patient is one thatis suitable for that patient. One of skill in the art would appreciatethat different individuals may require different total amounts of thetherapeutically effective substance. In some embodiments, the amount ofthe therapeutically effective substance is a pharmaceutically effectiveamount. The skilled worker would be able to determine the amount of thetherapeutically effective substance in a composition needed to treat apatient based on factors such as, for example, the age, weight, andphysical condition of the patient. The concentration of therapeuticallyeffective substance (e.g., DOXO-EMCH) depends on its solubility in theintravenous administration solution and the volume of fluid that can beadministered. For example, the concentration of the therapeuticallyeffective substance may be from about 0.1 mg/ml to about 50 mg/ml in theinjectable composition. In some embodiments, the concentration of thetherapeutically effective substance may be from about 0.1 mg/ml to about25 mg/ml, from about 7 mg/ml to about 17 mg/ml, from about 0.1 mg/ml toabout 5 mg/ml, or from about 0.25 mg/ml to about 4.5 mg/ml. Inparticular embodiments, the concentration of the therapeuticallyeffective substance may be about 0.1 mg/ml, about 0.2 mg/ml, about 0.3mg/ml, about 0.4 mg/ml, about 0.5 mg/ml, about 0.6 mg/ml, about 0.7mg/ml, about 0.8 mg/ml, about 0.9 mg/ml, about 1.0 mg/ml, about 1.1mg/ml, about 1.2 mg/ml, about 1.3 mg/ml, about 1.4 mg/ml, about 1.5mg/ml, about 1.6 mg/ml, about 1.7 mg/ml, about 1.8 mg/ml, about 1.9mg/ml, about 2.0 mg/ml, about 2.1 mg/ml, about 2.2 mg/ml, about 2.3mg/ml, about 2.4 mg/ml, about 2.5 mg/ml, about 2.6 mg/ml, about 2.7mg/ml, about 2.8 mg/ml, about 2.9 mg/ml, about 3.0 mg/ml, about 3.1mg/ml, about 3.2 mg/ml, about 3.3 mg/ml, about 3.4 mg/ml, about 3.5mg/ml, about 3.6 mg/ml, about 3.7 mg/ml, about 3.8 mg/ml, about 3.9mg/ml, about 4.0 mg/ml, about 4.1 mg/ml, about 4.2 mg/ml, about 4.3mg/ml, about 4.4 mg/ml, about 4.5 mg/ml, about 4.6 mg/ml, about 4.7mg/ml, about 4.8 mg/ml, about 4.9 mg/ml, about 5.0 mg/ml, about 5.1mg/ml, about 5.2 mg/ml, about 5.3 mg/ml, about 5.4 mg/ml, about 5.5mg/ml, about 5.6 mg/ml, about 5.7 mg/ml, about 5.8 mg/ml, about 5.9mg/ml, or about 6.0 mg/ml. In some embodiments, the concentration of thetherapeutically effective substance may be about 7 mg/ml, about 8 mg/ml,about 9 mg/ml, about 10 mg/ml, about 11 mg/ml, about 12 mg/ml, about 13mg/ml, about 14 mg/ml, about 15 mg/ml, about 16 mg/ml, about 17 mg/ml,about 18 mg/ml, about 19 mg/ml, about 20 mg/ml, about 21 mg/ml, about 22mg/ml, about 23 mg/ml, about 24 mg/ml, about 25 mg/ml, about 26 mg/ml,about 27 mg/ml, about 28 mg/ml, about 29 mg/ml, or about 30 mg/ml.

The pharmaceutical compositions and kits of the present invention mayalso contain diluents, fillers, salts, buffers, stabilizers,solubilizers, and other materials well known in the art.

The compositions may be administered in a variety of conventional ways.Exemplary routes of administration that can be used include oral,parenteral, intravenous, intra-arterial, cutaneous, subcutaneous,intramuscular, topical, intracranial, intraorbital, ophthalmic,intravitreal, intraventricular, intracapsular, intraspinal,intracisternal, intraperitoneal, intranasal, aerosol, central nervoussystem (CNS) administration, or administration by suppository. In someembodiments, the compositions are suitable for parenteraladministration. These compositions may be administered, for example,intraperitoneally, intravenously, or intrathecally. In some embodiments,the compositions are injected intravenously. In some embodiments, areconstituted formulation can be prepared by reconstituting alyophilized anthracycline compound composition in a reconstitutionliquid comprising ethanol and water. Such reconstitution may compriseadding the reconstitution liquid and mixing, for example, by swirling orvortexing the mixture. The reconstituted formulation then can be madesuitable for injection by mixing e.g., Lactated Ringer's solution withthe formulation to create an injectable composition. One of skill in theart would appreciate that a method of administering a therapeuticallyeffective substance formulation or composition would depend on factorssuch as the age, weight, and physical condition of the patient beingtreated, and the disease or condition being treated. The skilled workerwould, thus, be able to select a method of administration optimal for apatient on a case-by-case basis.

In some embodiments, the composition of the therapeutically effectivesubstance may be used in the manufacture of a medicament for treatingbrain cancer.

In some embodiments, the present invention provides a kit comprising atherapeutically effective substance as described herein and, apharmaceutically acceptable excipient, a carrier, and/or a diluent.

In some embodiments, one or more excipients may be included in thecomposition. One of skill in the art would appreciate that the choice ofany one excipient may influence the choice of any other excipient. Forexample, the choice of a particular excipient may preclude the use ofone or more additional excipients because the combination of excipientswould produce undesirable effects. One of skill in the art would be ableto empirically determine which excipients, if any, to include in thecompositions. Excipients may include, but are not limited to,co-solvents, solubilizing agents, buffers, pH adjusting agents, bulkingagents, surfactants, encapsulating agents, tonicity-adjusting agents,stabilizing agents, protectants, and viscosity modifiers. In someembodiments, it may be beneficial to include a pharmaceuticallyacceptable carrier in the compositions.

In some embodiments, a solubilizing agent may be included compositions.Solubilizing agents may be useful for increasing the solubility of anyof the components of the composition, including a therapeuticallyeffective substance (e.g., DOXO-EMCH) or an excipient. The solubilizingagents described herein are not intended to constitute an exhaustivelist, but are provided merely as exemplary solubilizing agents that maybe used in the compositions. In certain embodiments, solubilizing agentsinclude, but are not limited to, ethyl alcohol, tert-butyl alcohol,polyethylene glycol, glycerol, methylparaben, propylparaben,polyethylene glycol, polyvinyl pyrrolidone, and any pharmaceuticallyacceptable salts and/or combinations thereof.

The pH of the compositions may be any pH that provides desirableproperties for the formulation or composition. Desirable properties mayinclude, for example, therapeutically effective substance (e.g.,DOXO-EMCH) stability, increased therapeutically effective substanceretention as compared to compositions at other pHs, and improvedfiltration efficiency. In some embodiments, the pH of the compositionsmay be from about 3.0 to about 9.0, e.g., from about 5.0 to about 7.0.In particular embodiments, the pH of the compositions may be 5.5±0.1,5.6±0.1, 5.7±0.1, 5.8±0.1, 5.9±0.1, 6.0±0.1, 6.1±0.1, 6.2±0.1, 6.3±0.1,6.4±0.1, or 6.5±0.1.

In some embodiments, it may be beneficial to buffer the pH by includingone or more buffers in the compositions. In certain embodiments, abuffer may have a pKa of, for example, about 5.5, about 6.0, or about6.5. One of skill in the art would appreciate that an appropriate buffermay be chosen for inclusion in compositions based on its pKa and otherproperties. Buffers are well known in the art. Accordingly, the buffersdescribed herein are not intended to constitute an exhaustive list, butare provided merely as exemplary buffers that may be used in theformulations or compositions of the invention. In certain embodiments, abuffer includes, but is not limited to Tris, Tris HCl, potassiumphosphate, sodium phosphate, sodium citrate, sodium ascorbate,combinations of sodium and potassium phosphate, Tris/Tris HCl, sodiumbicarbonate, arginine phosphate, arginine hydrochloride, histidinehydrochloride, cacodylate, succinate, 2-(N-morpholino)ethanesulfonicacid (MES), maleate, bis-tris, phosphate, carbonate, and anypharmaceutically acceptable salts and/or combinations thereof.

In some embodiments, a pH-adjusting agent may be included in thecompositions. Modifying the pH of a composition may have beneficialeffects on, for example, the stability or solubility of atherapeutically effective substance, or may be useful in making acomposition suitable for parenteral administration. pH-adjusting agentsare well known in the art. Accordingly, the pH-adjusting agentsdescribed herein are not intended to constitute an exhaustive list, butare provided merely as exemplary pH-adjusting agents that may be used inthe compositions. pH-adjusting agents may include, for example, acidsand bases. In some embodiments, a pH-adjusting agent includes, but isnot limited to, acetic acid, hydrochloric acid, phosphoric acid, sodiumhydroxide, sodium carbonate, and combinations thereof.

In some embodiments, a bulking agent may be included in thecompositions. Bulking agents are commonly used in lyophilizedcompositions to provide added volume to the composition and to aidvisualization of the composition, especially in instances where thelyophilized pellet would otherwise be difficult to see. Bulking agentsalso may help prevent drug loss due to blowout of the activecomponent(s) of a pharmaceutical composition and/or to aidcryoprotection of the composition. Bulking agents are well known in theart. Accordingly, the bulking agents described herein are not intendedto constitute an exhaustive list, but are provided merely as exemplarybulking agents that may be used in the compositions.

Exemplary bulking agents may include carbohydrates, monosaccharides,disaccharides, polysaccharides, sugar alcohols, amino acids, and sugaracids, and combinations thereof. Carbohydrate bulking agents include,but are not limited to, mono-, di-, or poly-carbohydrates, starches,aldoses, ketoses, amino sugars, glyceraldehyde, arabinose, lyxose,pentose, ribose, xylose, galactose, glucose, hexose, idose, mannose,talose, heptose, glucose, fructose, methyl a-D-glucopyranoside, maltose,lactone, sorbose, erythrose, threose, arabinose, allose, altrose,gulose, idose, talose, erythrulose, ribulose, xylulose, psicose,tagatose, glucosamine, galactosamine, arabinans, fructans, fucans,galactans, galacturonans, glucans, mannans, xylans, inulin, levan,fucoidan, carrageenan, galactocarolose, pectins, amylose, pullulan,glycogen, amylopectin, cellulose, pustulan, chitin, agarose, keratin,chondroitin, dermatan, hyaluronic acid, xanthin gum, sucrose, trehalose,dextran, and lactose. Sugar alcohol bulking agents include, but are notlimited to, alditols, inositols, sorbitol, and mannitol. Amino acidbulking agents include, but are not limited to, glycine, histidine, andproline. Sugar acid bulking agents include, but are not limited to,aldonic acids, uronic acids, aldaric acids, gluconic acid, isoascorbicacid, ascorbic acid, glucaric acid, glucuronic acid, gluconic acid,glucaric acid, galacturonic acid, mannuronic acid, neuraminic acid,pectic acids, and alginic acid.

In some embodiments, a surfactant may be included in the compositions.Surfactants, in general, reduce the surface tension of a liquidcomposition. This may provide beneficial properties such as improvedease of filtration. Surfactants also may act as emulsifying agentsand/or solubilizing agents. Surfactants are well known in the art.Accordingly, the surfactants described herein are not intended toconstitute an exhaustive list, but are provided merely as exemplarysurfactants that may be used in the formulations or compositions of theinvention. Surfactants that may be included include, but are not limitedto, sorbitan esters such as polysorbates (e.g., polysorbate 20 andpolysorbate 80), lipopolysaccharides, polyethylene glycols (e.g., PEG400 and PEG 3000), poloxamers (i.e., pluronics), ethylene oxides andpolyethylene oxides (e.g., Triton X-100), saponins, phospholipids (e.g.,lecithin), and combinations thereof.

In some embodiments, an encapsulating agent may be included in thecompositions. Encapsulating agents can sequester molecules and helpstabilize or solubilize them. Encapsulating agents are well known in theart. Accordingly, the encapsulating agents described herein are notintended to constitute an exhaustive list, but are provided merely asexemplary encapsulating agents that may be used in the compositions.Encapsulating agents that may be included in compositions include, butare not limited to, dimethyl-β-cyclodextrin,hydroxyethyl-β-cyclodextrin, hydroxypropyl-β-cyclodextrin, andtrimethyl-β-cyclodextrin, and combinations thereof.

In some embodiments, a tonicity-adjusting agent may be included in thecompositions. The tonicity of a liquid composition is an importantconsideration when administering the composition to a patient, forexample, by parenteral administration. Tonicity-adjusting agents, thus,may be used to help make a composition suitable for administration.Tonicity-adjusting agents are well known in the art. Accordingly, thetonicity-adjusting agents described herein are not intended toconstitute an exhaustive list, but are provided merely as exemplarytonicity-adjusting agents that may be used in the compositions.Tonicity-adjusting agents may be ionic or non-ionic and include, but arenot limited to, inorganic salts, amino acids, carbohydrates, sugars,sugar alcohols, and carbohydrates. Exemplary inorganic salts may includesodium chloride, potassium chloride, sodium sulfate, and potassiumsulfate. An exemplary amino acid is glycine. Exemplary sugars mayinclude sugar alcohols such as glycerol, propylene glycol, glucose,sucrose, lactose, and mannitol.

In some embodiments, a stabilizing agent may be included in thecompositions. Stabilizing agents help increase the stability of atherapeutically effective substance in the compositions. This may occurby, for example, reducing degradation or preventing aggregation of atherapeutically effective substance. Without wishing to be bound bytheory, mechanisms for enhancing stability may include sequestration ofthe therapeutically effective substance from a solvent or inhibitingfree radical oxidation of the anthracycline compound. Stabilizing agentsare well known in the art. Accordingly, the stabilizing agents describedherein are not intended to constitute an exhaustive list, but areprovided merely as exemplary stabilizing agents that may be used in thecompositions. Stabilizing agents may include, but are not limited to,emulsifiers and surfactants.

In some embodiments, a protectant may be included in the compositions.Protectants are agents that protect a pharmaceutically active ingredient(e.g., a therapeutically effective substance) from an undesirablecondition (e.g., instability caused by freezing or lyophilization, oroxidation). Protectants can include, for example, cryoprotectants,lyoprotectants, and antioxidants. Cryoprotectants are useful inpreventing loss of potency of an active pharmaceutical ingredient (e.g.,an anthracycline compound) when a composition is exposed to atemperature below its freezing point. For example, a cryoprotectantcould be included in a reconstituted lyophilized formulation so that theformulation could be frozen before dilution for intravenous (IV)administration. Cryoprotectants are well known in the art. Accordingly,the cryoprotectants described herein are not intended to constitute anexhaustive list, but are provided merely as exemplary cryoprotectantsthat may be used in the compositions. Cryoprotectants include, but arenot limited to, solvents, surfactants, encapsulating agents, stabilizingagents, viscosity modifiers, and combinations thereof. Cryoprotectantsmay include, for example, disaccharides (e.g., sucrose, lactose,maltose, and trehalose), polyols (e.g., glycerol, mannitol, sorbitol,and dulcitol), glycols (e.g., ethylene glycol, polyethylene glycol andpropylene glycol).

Lyoprotectants are useful in stabilizing the components of acomposition. For example, a therapeutically effective substance could belyophilized with a lyoprotectant prior to reconstitution. Lyoprotectantsare well known in the art. Accordingly, the lyoprotectants describedherein are not intended to constitute an exhaustive list, but areprovided merely as exemplary lyoprotectants that may be used in thecompositions. Lyoprotectacts include, but are not limited to, solvents,surfactants, encapsulating agents, stabilizing agents, viscositymodifiers, and combinations thereof. Exemplary lyoprotectants may be,for example, sugars and polyols. Trehalose, sucrose, dextran, andhydroxypropyl-beta-cyclodextrin are non-limiting examples oflyoprotectants.

Antioxidants are useful in preventing oxidation of the components of acomposition. Oxidation may result in aggregation of a drug product orother detrimental effects to the purity of the drug product or itspotency. Antioxidants are well known in the art. Accordingly, theantioxidants described herein are not intended to constitute anexhaustive list, but are provided merely as exemplary antioxidants thatmay be used in the compositions. Antioxidants may be, for example,sodium ascorbate, citrate, thiols, metabisulfite, and combinationsthereof.

In some embodiments, a viscosity modifying agent may be included in thecomposition. Viscosity modifiers change the viscosity of liquidcompositions. This may be beneficial because viscosity plays animportant role in the ease with which a liquid composition is filtered.A composition may be filtered prior to lyophilization andreconstitution, or after reconstitution. Viscosity modifiers are wellknown in the art. Accordingly, the viscosity modifiers described hereinare not intended to constitute an exhaustive list, but are providedmerely as exemplary viscosity modifiers that may be used in thecompositions. Viscosity modifiers include solvents, solubilizing agents,surfactants, and encapsulating agents. Exemplary viscosity modifiersthat may be included in compositions include, but are not limited to,N-acetyl-DL-tryptophan and N-acetyl-cysteine.

Methods of Treatment

The methods of treatment provided herein are useful for a variety ofclinical applications. Anthracyclines are useful in the treatment ofcancer. For example, doxorubicin is an intercalating agent as well as atopoisomerase II inhibitor, and preferentially kills rapidly dividingcells, such as tumor cells. DOXO-EMCH is an anthracycline compound thatcan be used to treat solid tumors as well as hematological malignancies.DOXO-EMCH acts by covalently binding to albumin wherein the free thiolof cysteine-34 of albumin binds the DOXO-EMCH maleimide via a Michaeladdition. It is believed that DOXO-EMCH-albumin conjugate thencirculates in the bloodstream until reaching a tumor, where the lower pHin the tumor results in cleavage of the hydrazone bond betweendoxorubicin and the EMCH moiety, thereby releasing the doxorubicin.

In one aspect, the invention provides methods for treating brain cancer.In some embodiments, the cancer is a primary brain cancer. Examples of aprimary brain cancer include glioma, astrocytoma, oligodendroglioma,ependymoma, meningioma, craniopharyngioma, germinoma, pineocytoma,pineoblastoma and glioblastoma multiforme. In some embodiments, theprimary brain cancer is glioblastoma multiforme. In some embodiments,the cancer is a metastatic or secondary brain cancer. Examples ofmetastatic or secondary brain cancers include a solid tumor cancer,breast cancer, lung cancer, endometrial cancer, ovarian cancer,pancreatic cancer, pancreatic ductal adenocarcinoma, cancer of theadrenal cortex, non-Hodgkin's lymphoma, multiple myeloma, leukemia,Kaposi's sarcoma, Ewing's sarcoma, soft tissue sarcoma, nephroblastoma,glioblastoma multiforme, prostate cancer, liver cancer, bone cancer,chondrosarcoma, renal cancer, bladder cancer, and gastric cancer. Insome embodiments, the metastatic or secondary brain cancer isglioblastoma multiforme. In some embodiments, the cancer is atemozolomide-resistant cancer. In some embodiments, thetemozolomide-resistant cancer is a temozolomide-resistant glioblastomamultiforme.

In another aspect, the invention provides methods for treating a primarybrain tumor. In some embodiments, the primary brain tumor isglioblastoma multiforme. In other embodiments, the brain cancer is ametastatic brain tumor. In some embodiments, the metastatic tumor isfrom a cancer including but not limited to breast cancer, lung cancer,stomach cancer, endometrial cancer, ovarian cancer, pancreatic cancer,pancreatic ductal adenocarcinoma, cancer of the adrenal cortex,non-Hodgkin's lymphoma, multiple myeloma, leukemia, Kaposi's sarcoma,Ewing's sarcoma, soft tissue sarcoma, nephroblastoma, glioblastoma,prostate cancer, liver cancer, bone cancer, chondrosarcoma, renalcancer, bladder cancer, thyroid cancer and gastric cancer. In someembodiments, the tumor is a temozolomide-resistant tumor. In someembodiments, the temozolomide-resistant tumor is atemozolomide-resistant glioblastoma multiforme.

In some embodiments, the method comprises administering DOXO-EMCH (i.e.,aldoxorubicin) either alone or in combination with an anti-cancer agentfor treating cancers or tumors. In some embodiments, the methodcomprises administering DOXO-EMCH (i.e., aldoxorubicin) either alone orin combination with an anti-cancer agent for treatingtemozolomide-resistant tumors. In some embodiments, the method comprisesadministering DOXO-EMCH (i.e., aldoxorubicin) for treatingtemozolomide-resistant tumors or cancers. In some embodiments, themethod comprises administering DOXO-EMCH (i.e., aldoxorubicin) fortreating glioblastoma multiforme. In other embodiments, the methodcomprises administering DOXO-EMCH (i.e., aldoxorubicin) for treatingtemozolomide-resistant glioblastoma multiforme.

Variations and Modifications

Variations, modifications, and other implementations of what isdescribed herein will occur to those of ordinary skill without departingfrom the spirit and the scope of the invention. Accordingly, theinvention is not to be limited to the preceding description or thefollowing examples.

Exemplification

With aspects of the invention now being generally described, these willbe more readily understood by reference to the following examples, whichare included merely for purposes of illustration of certain features andembodiments of the invention and are not intended to be limiting.

Equivalents

Those skilled in the art will recognize, or be able to ascertain usingno more than routine experimentation, numerous equivalents to thecompounds, compositions, and methods of use thereof described herein.Such equivalents are considered to be within the scope of the invention.

EXAMPLES

The preclinical efficacy of doxorubicin versus aldoxorubicin wascompared in an in vivo mouse model for glioblastoma.

Example 1: Aldoxorubicin, but not Doxorubicin, Induces Tumor Regressionand Significantly Increases Survival in Xenograft Mouse Model

Intracranial implantation of U87-luc glioma cells in mice: A U87MGsubline, U87-luc with a luciferase reporter gene was used forestablishing intracranial human glioblastoma tumors. Female BALB/c(nu/nu) mice, 6-8 weeks of age, were anesthetized with aketamine/xylazine cocktail solution. Animals were secured in a HarvardApparatus stereotaxic head frame, a 1 cm midline scalp incision wasmade, and 5×10⁵ cells in 5 μL serum-free DMEM were injected into theleft striatum (coordinates: 2.5 mm lateral and 0.5 mm posterior to thebregma) through a burr hole in the skull using a 10 μl Hamilton syringeto deliver tumor cells to a 3.5 mm intraparenchymal depth. The burr holein the skull was sealed with bone wax and the incision closed usingwound glue. Tumor growth was evaluated by bioluminescent imaging.

Aldoxorubicin treatment of mice: The study consisted of 8vehicle-treated control mice (group C), 8 doxorubicin-treated mice(group D), and 8 aldoxorubicin-treated mice (group A). Treatment wasinitiated twelve days after intracranial implantation of glioblastomamultiforme (GBM) cells. Vehicle (10 mM sodium phosphate, 5%D-(+)-glucose, pH 6.4) or aldoxorubicin was administered intravenouslyfor a total of six injections (i.e., 12, 19, 26, 42, 50, and 56, daysafter cell implantation). All the doses were ˜75% of the maximumtolerated dose (MTD) of 32 mg/kg/inj in mice except that the dose givenafter 50 days of cell implantation was 50% of the MTD. Doxorubicin wasadministered intravenously for a total of two injections (i.e., 12 and19 days after cell implantation) with ˜75% of the MTD of 8 mg/kg/inj.Both the drugs and the vehicle were administered using an injectionvolume of 0.15 ml.

In vivo imaging of intracranial tumors: Intracranial tumor growth wasquantified by bioluminescent imaging using an in vivo imaging system(Xenogen, Palo Alto, Calif.). All mice were given an IP injection of 100μl of 30 mg/ml D-luciferin (PerkinElmer) suspended in DPBS 10 minutesbefore imaging to provide a substrate for the luciferase enzyme. Priorto imaging, mice were anesthetized with inhalation of isoflurane gas.Images were captured using the Xenogen Ivis 200 imaging system andquantified with Living Image 4.1 software from Xenogen for a region ofinterest that encompassed the head of the mouse. Image intensities wereexpressed as photons/sec/cm²/sr.

HPLC System and Conditions: An Agilent 1100 Series HPLC System(Wilmington, Del., USA) having a scanning fluorescent detector withexcitation and emission wavelengths set at 480 and 560 nm, respectively,was used. Agilent Chemstation software was used for data acquisition.Separation was achieved on a Waters Spherisorb ODS2 column (4 mm×250 mm,5 μm) fitted with a guard cartridge (BDS-Hypersil-C18, 5 μM). Elutionwas performed with mobile phase containing 65% monosodium phosphate, pH2.2, and 35% acetonitrile. A constant flow rate of 1.25 ml/min was usedfor the separation. The column was set to 28° C. and the injectionvolume was 25 μl.

Doxorubicin, aldoxorubicin, and the internal standard daunorubicindemonstrated average retention times of 4.06, 4.39 and 6.52 min.,respectively, and were sufficiently resolved under the applied assayconditions. In the organ samples analyzed, aldoxorubicin eluted with theretention time of doxorubicin. No interfering peaks were observed underthe chromatography conditions used.

Sample preparation: Aldoxorubicin, 24 mg/kg/inj (75% of the MTD), wasadministered in intracranial GBM tumor-bearing mice through tail veininjection, and after 4, 8, 16, and 24 h after injection, mice (3 animalsat each time point) were euthanized by CO₂ gas. Blood samples werecollected by heart puncture in heparinized tubes, and centrifuged forplasma separation. Immediately after blood sampling, organs (brain,heart, kidney liver and lung) were surgically removed. The plasma andtissues were stored at −80° C. until analysis.

Frozen samples were thawed at room temperature and homogenized insterile saline using a PowerGen Model 125 homogenizer (FisherScientific) to obtain final tissue concentrations (w/v) of 150 mg/ml forthe liver and brain; 125 mg/ml for the lung, heart and muscle; and 100mg/ml for kidney. Perchloric acid (35%, v/v) was added to a 20 μlaliquot of plasma or tissue samples followed by 25 μl of mobile phase.The samples were vortexed followed by centrifugation at 10,000×g for 10min. and 25 μl of the supernatant was applied to the HPLC column.

Statistical Analysis: The log-rank test was used to create Kaplan-Meiersurvival curves to compare survival between control and drug-treatedmice using the Statistical Analysis Software of SAS Institute, Inc.,Cary, N.C. Differences between groups were assessed using the unpairedStudent's t test. AR values are shown as the mean±standard deviation. Ap value≤0.05 was considered statistically significant.

Results

Aldoxorubicin but not doxorubicin is a potent inhibitor of glioma tumorsin mice. FIGS. 1 and 2 show that there was no relative difference inaverage tumor sizes between the control group (group C), doxorubicintreatment group (group D) and the aldoxorubicin treatment group (groupA) after 8 days of intracranial tumor cell implantation (p>0.05). 3animals in the control group and 2 in the doxorubicin group died andothers developed large tumors in 22 days, Aldoxorubicin treatment for 2weeks showed tumor regression resulting in average tumor size of 28%that of the control group and 40% that of doxorubicin treatment group(FIG. 2D). Further, animals in the control and doxorubicin treatmentgroups experienced much shorter survival compared to animals in thealdoxorubicin treatment group. All animals in the control group (groupC) and doxorubicin group (group D) died within 34 days after tumorimplantation, but animals in the treatment group (group A) remainedalive. Even after 41 days, seven of the eight animals in thealdoxorubicin group were still alive. See FIG. 1.

FIG. 3 features Kaplan-Meier survival curves showing increased survivaltimes (p<0.0001) in mice treated with aldoxorubicin as compared with thevehicle-treated or the doxorubicin-treated group. There was nodifference in the survival time between the vehicle-treated and thedoxorubicin-treated mice (p=0.949). When the study was terminated, thesurviving animals were censored because they had not reached theendpoint.

HPLC was used to determine the plasma and tissue distribution ofaldoxorubicin its administration to intracranial tumor-bearing mice. Theconcentration vs. time profile is shown in FIG. 4. After intravenousadministration of aldoxorubicin (˜75% of the MTD), the drugconcentrations were the highest after 4 h in plasma and other organsexcept brain. Plasma concentration was more than 20 fold higher than themean concentration in liver, heart, lung and kidney, and more than 200fold higher when compared to that in the brain. The prodrug reachednearly 50% of its concentration in 20 h in plasma and in other tissuesexcept in the brain. In the brain, the levels remained almost same from4 h to 24 h, suggesting that the high antitumor activity in brain may beassociated with prolonged presence of the drug.

Conclusions

These results demonstrate that aldoxorubicin, but not doxorubicin,administered intravenously induces tumor regression and significantlyincreases survival in an in vivo xenograft model employing intracranialimplantation of human GBM tumors.

Example 2: Aldoxorubicin-Induced Tumor Regression and Increase ofSurvival in Xenograft Mouse Model

Female mice (6-8 weeks of age) were implanted intracranially withU87-luc subline with luciferase reporter gene to establish humanglioblastoma tumors. Tumor growth was evaluated by bioluminescentimaging using D-luciferin substrate. 8 mice were treated withaldoxorubicin and 8 were treated with phosphate buffer saline (vehicle).Treatment started 9 days after implantation of the GBM cells.Aldoxorubicin or vehicle was administered i.v. once a week for threeweeks (9, 16, and 23 days after cell implantation). The first two dosesof aldoxorubicin were 75% and the third dose was 50% of the MTD of 32mg/kg/injection in mice. Results are shown in FIG. 5.

Conclusions

These results demonstrate that aldoxorubicin administered intravenouslyinduces tumor regression and increases survival in an in vivo xenograftmodel employing intracranial implantation of human GBM tumors.

Example 3: Aldoxorubicin Retention in Tumor Tissues

HPLC System and Conditions: The HPLC system used was an Agilent 1100Series (Wilmington, Del., USA) equipped with a scanning fluorescentdetector with excitation and emission wavelengths set at 480 and 560 nm,respectively. Agilent Chemstation software was used for dataacquisition. Separation was achieved on a Waters Spherisorb ODS2 column(4 mm×250 mm, 5 μm) fitted with a guard cartridge (BDS-Hypersil-C18, 5μM). Elution was performed with mobile phase comprised of 65% 50 mMmonosodium phosphate, pH 2.2, and 35% acetonitrile. A constant flow rateof 1.25 ml/min was used for the separation. The column was set to 28° C.and the injection volume was 25 μl.

Doxorubicin, aldoxorubicin, and the internal standard daunorubicindemonstrated average retention times of 4.06, 4.39 and 6.52 min,respectively, and were sufficiently resolved under the applied assayconditions. In the organ samples analyzed, aldoxorubicin eluted with theretention time of doxorubicin. No interfering peaks were observed underthe chromatography conditions used.

Sample preparation: For quantification of aldoxorubicin in brain tissueand brain tumors, mice were euthanized by CO₂ inhalation 6 and 24 hafter aldoxorubicin injection (24 mg/kg/inj), brains were harvested andtumors were resected. The harvested tissues were stored at −80° C. untilanalysis.

Frozen samples were thawed at room temperature and homogenized insterile saline using a PowerGen Model 125 homogenizer (FisherScientific) to obtain final tissue concentrations (w/v) of 150 mg/ml.Perchloric acid (35%, v/v) was then added to a 2.0 μl aliquot followedby 25 μl of mobile phase. The samples were vortexed followed bycentrifugation at 10,000×g for 10 min and 2.5 μl of the supernatant wasapplied to the HPLC column. In the tissue samples analyzed,aldoxorubicin eluted with the retention time of doxorubicin.

Conclusions

Aldoxorubicin retention was 3- to 4-fold higher in tumor tissues than inthe surrounding brain tissues. See FIG. 8.

Example 4: Short Description of the Experiment

Immunohistochemistry: For histologic analysis, brain tissues fromcontrol and drug-treated tumor-bearing mice were harvested, snap frozenin optimal cutting temperature (OCT) compound and stored at −80° C.Cryostat sections were placed on slides and fixed in zinc-bufferedformalin. Slides were blocked with 5% goat serum in 1% BSA followed byovernight incubation with primary antibodies against CD31 (102402,Biolegend, San Diego, Calif.), Ki-67 (ab156956, Abcam, Cambridge,Mass.), Vimentin (ab92547, Abcam), cleaved-Caspase-3 (CP229B, BiocareMedical), and GFAP (NB300-141, Novus Biologicals, Littleton, Colo.).Slides were then incubated with primary antibody source-specificsecondary antibodies conjugated to Alexa Fluor 488 or 635 and DAN as anuclear counterstain. The detection fluorophores used were limited tothose around the inherent fluorescence spectra of doxorubicin(λ_(ex)=480 nm, λ_(em)=550-590 nm) (22876313) to avoid bleed-through andenable co-detection of the drug with respect to certain antigens.Epifluorescence photomicrographs were captured at 100× and 400×magnification using an FV1000 confocal microscope (Olympus of America,Center Valley, Pa.) equipped with multi-Argon, 405, 559, and 635 diodes.Quantitative analysis was performed with Slidebook software (IntelligentImaging Innovations, Denver, Colo.). FIGS. 9 and 11 illustrate thequantitative analyses of the data obtained by immunohistochemicalanalysis.

Conclusions

Aldoxorubicin accumulates in the brain tumor but not in normal braintissue. Doxorubicin is not found in any appreciable amount in either thetumor or normal brain.

Example 5: Short Description of the Experiment

Aldoxorubicin/doxorubicin detection in brain tumors: Tumor-bearing micewere given intravenous injections of aldoxorubicin or doxorubicin asdescribed above in Example 1. Mice were euthanized 24 h following thelast injection. Brains were harvested and imaged using an MVX10stereomicroscope (Olympus of America) equipped for brightfield andepifluorescence with filters encompassing doxorubicin-specificwavelengths to visualize drug accumulation. See FIG. 10.

Conclusions

Aldoxorubicin and not doxorubicin accumulates in glioblastoma tumors.

The invention claimed is:
 1. A pharmaceutical composition comprising atherapeutically effective substance for use in the treatment of braincancer, wherein the therapeutically effective substance comprisesDOXO-EMCH or a pharmaceutically acceptable salt thereof, wherein thetherapeutically effective substance binds to serum albumin, and whereinthe composition further comprises sodium bicarbonate and tert-butylalcohol.
 2. The pharmaceutical composition according to claim 1, whereinthe brain cancer is a primary brain cancer.
 3. The pharmaceuticalcomposition according to claim 2, wherein the primary brain cancer isglioma, astrocytoma, oligodendroglioma, ependymoma, meningioma,craniopharyngioma, germinoma, pineocytoma, pineoblastoma andglioblastoma multiforme.
 4. The pharmaceutical composition according toclaim 3, wherein the primary brain cancer is glioblastoma multiforme. 5.The pharmaceutical composition according to claim 1, wherein the braincancer is a secondary or metastatic cancer.
 6. The pharmaceuticalcomposition according to claim 5, wherein the secondary or metastaticcancer is selected from bladder cancer, breast cancer, lung cancer,stomach cancer, endometrial cancer, ovarian cancer, pancreatic cancer,pancreatic ductal adenocarcinoma, cancer of the adrenal cortex,non-Hodgkin's lymphoma, multiple myeloma, leukemia, Kaposi's sarcoma,Ewing's sarcoma, soft tissue sarcoma, nephroblastoma, prostate cancer,liver cancer, bone cancer, chondrosarcoma, renal cancer, bladder cancer,thyroid cancer and gastric cancer.
 7. The pharmaceutical compositionaccording to claim 1, wherein the composition further comprises ananti-cancer agent.
 8. The pharmaceutical composition according to claim7, wherein the anti-cancer agent is selected from doxorubicin,cisplatin, carboplatin, paclitaxel, docetaxel, temozolomide,nitrosoureas, bortezomib, gemcitabine, etoposide, topotecan, or apharmaceutically acceptable salt thereof.
 9. The pharmaceuticalcomposition according to claim 1, wherein the therapeutically effectivesubstance is(E)-N′-(1-((2S,4S)-4-(4-amino-5-hydroxy-6-methyl-tetrahydro-2H-pyran-2-yloxy)-2,5,12-trihydroxy-7-methoxy-6,11-dioxo-1,2,3,4,6,11-hexahydrotetracen-2-yl)-2-hydroxyethylidene)-6-(2,5-dioxo-2H-pyrrol-1(5H)yl)hexanehydrazide.HCl.